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Colloidal gold test strip for rapidly detecting double viruses of tobacco mosaic virus-potato virus Y (TMV-PVY)

A colloidal gold and virus technology, which is applied in the field of pathogen detection, can solve the problems of being expensive and unsuitable for tobacco seedling detection and use, and achieves the effects of simple operation, easy detection of a large number of samples, and convenient operation.

Inactive Publication Date: 2016-12-21
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Foreign commercial test strips are very expensive, the price is 50-60 yuan / sheet, which is not suitable for the detection of a large number of tobacco seedlings in production

Method used

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  • Colloidal gold test strip for rapidly detecting double viruses of tobacco mosaic virus-potato virus Y (TMV-PVY)
  • Colloidal gold test strip for rapidly detecting double viruses of tobacco mosaic virus-potato virus Y (TMV-PVY)
  • Colloidal gold test strip for rapidly detecting double viruses of tobacco mosaic virus-potato virus Y (TMV-PVY)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The isolation of TMV, PVY virus strain of embodiment 1 Guangdong smoking area

[0039] 1. From 2012 to 2014, from Nanxiong, Shixing, Ruyuan, and Lechang in Shaoguan City, Guangdong Province, Lianzhou in Qingyuan City, Wuhua, Jiaoling, Dapu and Meixian in Meizhou City, etc. 29 Sampling was carried out on tobacco plants exhibiting common mosaic disease in tobacco fields in 3 towns and 33 villages. The sampling points covered all smoking areas in Guangdong Province. A total of 72 samples. Separate and identify them by biology and ELISA (enzyme-linked immunosorbent assay).

[0040] A total of 49 TMV isolates and 13 PVY isolates were isolated, all of which have been verified by ELISA. The results of biological identification showed that the collected TMV and PVY isolates belonged to their respective common strains.

[0041] 2. Amplify the full CP (coat protein) genes of the two viruses respectively. After the gel recovery of the PCR product, it was ligated with the pMD-...

Embodiment 2

[0045] Embodiment 2 prepares two kinds of CP gene prokaryotic expression specific proteins

[0046] CP-T-GD and CP-P-GD were respectively constructed on the pET30a-GST vector, transformed into BL21 strain, prokaryotic expression was carried out, and the expressed proteins were purified respectively.

[0047] 1. Preparation of CP-T-GD prokaryotic expression protein, relevant information is summarized in Table 1 below.

[0048] Table 1

[0049]

[0050] The specific method is as follows.

[0051] (1) Target gene sequence: according to the codon-optimized sequence of Escherichia coli, enzyme cutting sites EcoR I and Xho I are added at both ends. Synthesize the target gene into pUC57 vector.

[0052] (2) Connect the target gene to the pET30a-GST vector by enzyme digestion and ligation. The schematic diagram of the constructed recombinant vector is shown in the attached figure 1 shown.

[0053] Digestion of gene fragments: 43 μl recombinant plasmid, 1 μl EcoR I, 1 μl Xho I,...

Embodiment 3

[0094] Example 3 Construction of hybridoma cell lines expressing TMV and PVY two kinds of virus-specific monoclonal antibodies

[0095] Using the prokaryotic expression of specific proteins of the two CP genes obtained in Example 2, mice were immunized with the corresponding expressed proteins; a fusion test was carried out to obtain positive hybridoma cell lines producing specific monoclonal antibodies of the two viruses respectively.

[0096] 1. Construct a hybridoma cell line expressing TMV virus-specific monoclonal antibody, the method is shown in Table 3.

[0097] table 3

[0098]

[0099]

[0100] The specific method is as follows:

[0101] (1) immunity

[0102] 1) Initially immunize 4 SPF BALB / c female mice subcutaneously with "TMV" at the amount of 60ug protein / mouse, numbered as: 1, 2, 3, 4.

[0103] 2) Two weeks after the initial immunization, the subcutaneous booster immunization was given for the first time, with an immune dose of 30ug protein per mouse. ...

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Abstract

The invention discloses a colloidal gold test strip for rapidly detecting double viruses of tobacco mosaic virus-potato virus Y (TMV-PVY). The test strip consists of a sample pad, a colloidal gold pad, an NC film, a piece of water absorption filter paper and a back lining, wherein the colloidal gold pad is enveloped with a TMV specific antibody and a PVY specific antibody which are marked by colloidal gold, and the TMV specific antibody and the PVY specific antibody are respectively generated by secretion by hybridoma cell lines, i.e., BALB / c-15-50 and BALB / c-15-8; the NC film is provided with a detection line and a control line; the detection line is enveloped with specific antigens of the TMV and the PVY; the control line is enveloped with a second antibody marked by the colloidal gold. The rapid detection colloidal gold test strip is especially suitable for the detection on the TMV and the PVY in Guangdong tobacco-growing areas, and is rapid, sensitive and accurate in dection, low in cost and simple and convenient in operation; the test strip can be used to diagnose the two viruses at the same time by one-time sampling, thus being very suitable for field primary screening of a great batch of samples; therefore, the colloidal gold test strip has a good application value in actual production and is good in popularization and application prospects.

Description

technical field [0001] The invention belongs to the technical field of pathogen detection. More specifically, it relates to a TMV-PVY double virus colloidal gold rapid detection test strip. Background technique [0002] Tobacco mosaic virus (TMV) and potato virus Y (PVY) are the main viruses infecting tobacco. The viral diseases they cause cause huge economic losses to the tobacco industry every year. Rapid and accurate qualitative detection of related viruses is one of the important means to prevent and control diseases and reduce losses in production. [0003] In scientific research, the detection methods of plant viruses mainly include biological detection methods (discrimination of symptom types, identification of hosts, etc.), electron microscope technology, serological detection methods (enzyme linked immunosorbent assay (ELISA), etc.) and molecular Biological detection methods (including PCR technology, nucleic acid probe technology, etc.), etc. Although the isola...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/10G01N33/577G01N33/569G01N33/558C12R1/91
CPCC07K16/10G01N33/558G01N33/56983G01N33/577
Inventor 阮小蕾
Owner SOUTH CHINA AGRI UNIV
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